Trypanosomiasis, malaria, and leishmaniasis are major parasitic diseases in developing countries (McKerrow, J. H. et al., Annu. Rev. Microbiol. 47:821-853 (1993)). American trypanosomiasis, or Chagas' disease, is the leading cause of heart disease in Latin America (Libow, L. F. et al., Cutis, 48:37-40 (1991)). At least 16-18 million people are infected with Trypanosoma cruzi, resulting in more than 50,000 deaths each year (Godal, T. et al., J. Tropical diseases. WHO Division of Control in Tropical Diseases World Health Organization: Geneva, Switzerland, pp 12-13. (1990); World Health Organization website: http://www.who.int/ctd/html/chagburtre.html). The statistics for malaria are more sobering, with about 300-500 million clinical cases and about 3 million deaths each year. Further, at least 10 million people are infected with a form of Leishmania each year (see Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed, 1996, McGraw-Hill, New York).
Chagas' disease is transmitted to humans by blood-sucking triatomine vectors with an infectious trypomastigote form of the protozoan parasite T. cruzi (Bonaldo, M. C. et al., Exp. Parasitol, 73:44-51 (1981); Harth, G., et al., T. Cruzi. Mol. Biochem Parasitol, 58:17-24 (1993); Meirelles, M. N. L., et al., Mol. Biochem. Parasitol, 52:175-184 (1992)). African trypanosomiasis is transmitted to humans and cattle by tsetse flies and is caused by subspecies of T. brucei. So called “African sleeping sickness” is transmitted by an infectious trypomastigote from T. brucei gambiense, and T. brucei rhodesiense produces a progressive and usually fatal form of disease marked by early involvement of the central nervous system. T. brucei is further the cause of nagana in cattle, but bovine trypanosomiasis is also transmitted by T. congolense and T. evansi. In trypanosomiasis infections, the trypomastigote enters the host bloodstream and ultimately invades a cardiac muscle cell, where it transforms into the intracellular amastigote. The parasite may also be found in the blood, lymph, spinal fluid and cells of the gastrointestinal tract. Amastigotes replicate within cells, transform back to trypomastigotes, and rupture the cell, releasing the infectious form back into the bloodstream and other cells, amplifying the infection. Reviews of the current understanding and treatment of African and American trypanosomiasis infections is provided by Urbina (Curr Pharm Des (2002) 8:287) and Burchmore, et al (Curr Pharm Des (2002) 8:256).
Cruzain (aka cruzipain) is the major cysteine protease of T. cruzi. The protease is expressed in all life cycle stages of the parasite, but delivered to different cellular compartments in each stage. In the epimastigote stage, which occurs in the insect vector, the protease is in a lysosomal compartment where it functions to degrade proteins endocystosed from the insect gut. In the infectious trypomastigote stage, the protease appears at the flagellar pocket, the site of endocytosis and secretion. In the amastigote stage, within the mammalian host cell, the protease is both in the lysosomal compartment and on the surface of the parasite where it may function in nutrition, remodeling of the mammalian cell, or evasion of host defense mechanisms. Addition of a cruzain inhibitor such as Z-Phe-Ala-FMK (benzyloxy-carbonyl-L-phenylalanyl L-alanine fluoromethyl ketone) to cultures of mammalian cells exposed to trypomastigotes or to mammalian cells already infected with T. cruzi amastigotes blocks replication and differentiation of the parasite (Bonaldo, M. C. et al., Exp. Parasitol, 73:44-51 (1981); Harth, G., et al., T. Cruzi Mol. Biochem Parasitol, 58:17-24 (1993); Meirelles, M. N. L., et al., Mol. Biochem. Parasitol, 52:175-184 (1992)), thus arresting the parasite life cycle. Therefore, cruzain is essential for replication of the intracellular parasite. Treatment of T. cruzi-infected mice with a vinyl sulfone-derivatized pseudopeptide inhibitor of cruzain, N-methyl piperazine-Phe-homoPhe-vinyl sulfone phenyl, has resulted in a cure in a mouse model study (Engel, J. C. et al., J. Exp. Med., 188:725-734 (1998)). Thus, cruzain is an appealing target for new antitrypanosomal chemotherapy (McKerrow, J. H. et al., Bioorg. Med. Chem., 7:639-644 (1999)).
Malaria is caused by protozoa of the genus Plasmodium and is transmitted to humans through the bite of an infected anopheline mosquito. The parasites develop into tissue schizonts in hepatic parenchymal cells, and then are released into the circulation as merozoites, which invade erythrocytes. In erythrocytes, the merozoites mature from trophozoites into schizonts. Schizont-containing erythrocytes rupture to release merozoites that then invade more erythrocytes to continue the malarial cycle. Current understanding and treatment of plasmodium infections is reviewed in Winstanley (Lancet Infect Dis (2001) 1:206), Wongsrichanalai, et al (Lancet Infect Dis (2002) 2:209) and throughout the Feb. 7, 2002 issue of Nature (Lond) (vol. 415, issue 6872).
The majority of malaria infections is caused by Plasmodium falciparum (see Goodman & Gilman's The Pharmacological Basis of Therapeutics, supra). Papain-family cysteine proteases, known as falcipains, are hemoglobinases from P. falciparum that are essential to plasmodium trophozoite protein synthesis and development (Sijwali, et al (2001) Biochem J 360:481). Sequencing of the Plasmodium genome has revealed at least three falcipain cysteine proteases, designated falcipain-1, falcipain-2 and falcipain-3, where falcipain-2 and falcipain-3 are understood to account for the majority of hemoglobinase activity in the plasmodium trophozoite (Joachimiak, et al (2001) Mol. Med 7:698). The falcipains are homologous to cruzain (Venturini, et al (2000) Biochem Biophys Res Commun 270:437 and Selzer, et al (1997) Exp Parasitol 87:212) and at least the falcipain-2 sequence is highly conserved amongst different Plasmodium strains with different sensitivities to established antimalarial drugs (Singh and Rosenthal (2001) Antimicrob Agents Chemother 45:949). In in vitro studies, cysteine protease inhibitors blocked globin hydrolysis in Plasmodium infected erythrocytes (Rosenthal (1995) Exp. Parasitol 80:272 and Semenov et al (1998) Antimicrob Agents Chemother 42:2254). Importantly, oral or parenteral administration of fluoromethyl ketone or vinyl sulfone peptidyl inhibitors of falcipain cured treated mice that were infected with Plasmodium (Olson, et al (1999) Bioorg Med Chem 7:633). Therefore, the falcipains and other homologous cysteine proteases are also important antimalarial chemotherapeutic targets.
Leishmaniasis is caused by protozoal species and subspecies of Leishmania transmitted to humans by the bites of infected female phlebotamine sandflies. Promastigotes injected into the host are phagocytized by tissue monocytes and are transformed into amastigotes, which reside in intracellular phagolysosomes. Human leishmaniasis is classified into cutaneous, mucocutaneous and visceral (kala azar) forms. Reviews of the current understanding and chemotherapy of leishmaniasis is provided by Croft and Yardley (Curr Pharm Des (2002) 8:319), Kafetzis, et al (Curr Opin Infect Dis (2002) 15:289, and Hepburn (Curr Opin Infect Dis 14:151).
In vitro and in vivo studies also have demonstrated that cysteine protease inhibitors disrupt the infectious life cycle of Leishmania (see, Selzer, et al (1999) Proc Natl Acad Sci 96:11015; Das, et al (2001) J. Immunol 166:4020 and Salvati, et al (2001) Biochim Biophys Acta 1545:357). Like Trypanosoma and Plasmodium, Leishmania synthesize cathepsin-L-like cysteine proteases that are essential to their pathogenicity (Selzer, et al (1997) Exp Parasitol 87:212). The substrate recognition of one cysteine protease of L. mexicana, named CPB2.8 Delta CTE, has been demonstrated to be similar to the substrate preferences of cruzain (Alves, et al (2001) Mol Biochem Parasitol 117:137 and Alves, et al (2001) Mol Biochem Parasitol 116:1). Additionally, cruzain shares sequence similarity with homologous cysteine proteases from L. pifanoi, L. mexicana, and L. major (see Mottram, et al (1992) Mol Microbiol 6:1925, Rafati, et al (2001) Mol Biochem Parasitol 113:35 and GenBank numbers L29168, X62163 and AJ130942). Therefore, cysteine proteases also represent a potential chemotherapeutic target against Leishmania infections.
Drugs currently used in the treatment of trypanosomiasis include Nifurtimox, Benznidazole, Suramin, Pentamidine isethionate, Eflornithine and Melarsoprol. Current chemotherapeutics for the treatment of leishmaniasis include Stibogluconate sodium, Amphotericin B, and Pentamidine isethionate. Drugs used in the treatment of malaria include chloroquine phosphate, mefloquine, halofantrine, and quinidine gluconate in combination with an antifolate or an antibiotic. Although these protozoans are inhibited to some extent by the administration of available chemotherapeutics, the currently prescribed pharmacological compounds to counteract trypanosomiasis, malaria, and leishmaniasis are limited by the ability of the parasites to develop resistance to them and by their significant toxicity to the infected host. Therefore, there is an interest in developing new drugs that will interfere with the infectious life cycle of a parasite. Because cysteine proteases are essential to the life cycle of the parasites that cause trypanosomiasis, malaria and leishmaniasis, they are a logical target for newly developed chemotherapeutics (reviewed in Sajid and McKerrow, Mol Biochem Parasitol (2002) 120:1).
Several irreversible peptide-based inhibitor series including halomethyl ketones, diazomethanes, epoxysuccinyl derivatives, and vinyl sulfone derivatives targeting cysteine proteases have been developed (Otto, H. et al., Chem. Rev., 97:133-171 (1997)). A disadvantage of the chloromethyl ketones is their high reactivity and consequent lack of selectivity. They react with serine proteases and other SH-containing molecules, such as glutathione or nonproteolytic enzymes, and result in toxicity in vivo. To increase selectivity and reduce reactivity and toxicity, a less reactive series of compounds, including monofluoro methyl ketones, epoxy derivatives (Roush, W. R. et al., Tetrahedron, 56:9747-9762 (2000)), and vinyl sulfone derivatives (Bromme, D. et al., Biochem. J., 315:85-89 (1996); Palmer, J. T. et al., J. Med Chem. 38:3193-3196 (1995); Roush, W. R. et al., J. Am. Chem. Soc. 120:10994-10995 (1998)) were developed. However, the low oral bioavailability associated with peptidyl inhibitors makes the further pursuit of effective chemical compounds of great interest.
Pharmaceutical compounds having a semicarbazone scaffold have been evaluated for clinical use as an antihypertensive (Warren, J. D. et al., J. Med. Chem., 20:1520-1521 (1977)), anticonvulsant (Dimmock, J. R. et al., Epilepsia, 35:648-655 (1994); Pandeya, S. N. et al., Pharmacol Res., 37:17-22 (1998); Dimmock, J. R. et al., Drug Dev Res., 46:112-125 (1999)), and antiallodynic agent (Carter, R. B. et al., Proceeding, International Symposium “Ion Channels in Pain and Neuroprotection” March 14-17, San Francisco, Calif.; p 19 (1999)). For example, the semicarbazone compound 4-[4-fluorophenoxy]benzaldehyde semicarbazone has entered clinical trials for the treatment of neuropathic pain (Ramu, K. et al., Drug Metab. Dispos., 28:1153-1161 (2000)). Recently, 5-nitrofurfural N-butyl semicarbazone (Cerecetto. H. et al., Farmaco, 53:89-94 (1998); Cerecetto, H. et al., J. Med. Chem., 42:1941-1950 (1999); Cerecetto, H. et al., Eur. J. Med. Chem., 35:343-350 (2000)) has been shown to have antitrypanosomal activities targeting trypanothione reductase through a nitro anion radical mechanism, however, no clear target validation was reported in these papers.
Therefore, there is a pressing interest in developing potent, efficacious, economically synthesized pharmaceutical compounds with minimal toxicity and maximal bioavailability for the effective treatment of these infectious parasitic diseases.